Methods Of Treating Cancer Using Compounds Containing A Vascular Disrupting Agent

ABSTRACT

The present invention relates to prodrugs of vascular disrupting agents comprising a vascular disrupting agent (VDA) associated with a MMP proteolytic cleavage site and to the use of such prodrugs in the targeted treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/553,803 filed onNov. 25, 2014, which is a continuation of U.S. Ser. No. 13/125,732 filedon July 12, 201, which is a U.S. National Phase Application of PCTPatent Application No. PCT/GB2009/002484, filed Oct. 20, 2009, whichclaims priority to GB Patent Application No. 0819287.4, filed Oct. 22,2008, which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to prodrugs of vascular disrupting agentsand to the use of such compounds in the targeted treatment of cancer.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the computer readable form of the Sequence Listing filedin the application and 3 KB in size, is hereby incorporated by referencein its entirety.

BACKGROUND TO THE INVENTION

Targeting tumour vasculature as a molecular approach to cancerchemotherapies is becoming one of the highest scientific priorities. Twodrug models are emerging, i.e. one that prevents the formation of newblood vessels in the tumour (antiangiogenesis) and one that targetsvascular destruction.

Disruption of even a small proportion of the tumour vasculature has beendemonstrated to induce wide ranging tumour death and retardation ofmetastasis, since a single blood vessel is responsible for supportingthe survival of many tumour cells. Endothelial cells, which form themajor component of the vasculature, are highly dependent upon thetubulin cytoskeleton for their motility, invasion, attachment, alignmentand proliferation (Denekamp, J, Br J Cancer, 45, 136-139 (1982)). Agentswhich disrupt the endothelial microtubule network will therefore cause arapid collapse in tumour blood flow and a prolonged period of vascularshutdown, culminating in extensive tumour-cell necrosis (Tozer et al.,Nat Rev Cancer, 5, 423-435 (2005), Lippert J W, Bioorg Med Chem, 15,605-615 (2007)).

One of the most potent classes of cancer therapeutic drugs are thevascular disrupting agents (VDAs) which characteristically have good invitro cell cytotoxicities but often show poor specificity for killingtumour over normal tissues in vivo. Furthermore, many VDAs such as thetubulin binding agents are water insoluble and require formulationbefore evaluation in the clinic. The present invention aims to addressthe aforementioned problems.

Colchicine and its analogues are potent VDAs causing haemorrhage andsubsequent extensive necrosis in tumours (Tozer et al., Nat Rev Cancer,5, 423-435 (2005)), as a direct consequence of tubulin binding andinduction of microtubule depolymerisation (Chaudri et al., J Mol Biol,303, 679-692 (2000)). Colchicine has not, however, shown intrinsic valueas a clinically applicable anticancer therapeutic due to a high level oftoxicity and consequent very narrow therapeutic index (Tozer et al., NatRev Cancer, 5, 423-435 (2005); Quinn et al., J Med Chem, 24, 636-639(1981)). It would be desirable, therefore, to be able to target a VDAsuch as colchicine selectively to a tumour.

The present inventors have developed a system for overcoming the toxiceffect of systemic administration of potent anti-cancer agents inparticular vascular disrupting agents.

SUMMARY OF THE INVENTION

The present invention relates to prodrugs of vascular disrupting agentscomprising a vascular disrupting agent (VDA) associated with a MMPproteolytic cleavage site and to the use of such prodrugs in thetargeted treatment of cancer

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only withreference to the following Figures in which:

FIG. 1 is a graph showing the metabolism of prodrug-1 versus time intumour and non-tumour tissues ex vivo;

FIG. 2 is a graph showing accumulation and levels of prodrug-1 intumour-bearing mice following intraperitoneal administration;

FIG. 3 is a graph demonstrating levels of VDA accumulating followingintraperitoneal administration of prodrug-1 to tumour bearing mice;

FIG. 4 is a graph showing differential metabolism of prodrug-2 in tumourhomogenates expressing high MT1-MMP levels (HT1080) versus tumourhomogenates expressing low MT1-MMP levels (MCF-7);

FIG. 5 is a graph showing accumulation and levels of prodrug-2 in HT1080tumour bearing mice following intraperitoneal administration;

FIG. 6 is a graph demonstrating levels of VDA accumulating followingintraperitoneal administration of prodrug-2 to HT1080 tumour bearingmice.

FIG. 7: Relative mouse bodyweights during dose escalation study

FIG. 8: Tumour growth during treatment with ICT-2522 (warhead)

FIG. 9: Tumour growth during treatment with ICT-2588 (prodrug)

FIG. 10A-FIG. 10C Tumour growth curves for mice treated with pro-drug(ICT-2588) compared with warhead (ICT-2552) administered at the molarequivalent dose FIG. 10A: ICT-2588, 37.5 mg/kg & ICT-2552, 7.5 mg/kg;FIG. 10B: ICT-2588, 50.0 mg/kg & ICT-2552, 10.0 mg/kg; FIG. 10C:ICT-2588, 62.5 mg/kg & ICT-2552, 12.5 mg/kg; and FIG. 10D: ICT-2588,75.0 mg/kg & ICT-2552, 15.0 mg/kg;

FIG. 1l Pro-drug, ICT-2588, showing modifications includingphosphorylated amino acid residues, end-cap, warhead and P2′alternatives;

FIG. 12 A graph demonstrating the stability of ICT 3053 (P2′=Ala) inboth tumour (HT1080) and liver (murine) homogenates. This is an exampleof the data obtained for this series of molecules and a summary of thedata is given in table 1; and

FIG. 13A-FIG. 13D Stability data for prodrug (ICT2588) (FIG. 13A)molecules with phosphate attached to serine (ICT3047) (FIG. 13B)tyrosine (ICT 3048) (FIG. 13D) and both serine and tyrosine (ICT3028)(FIG. 13C). Data show percent of parent molecule remaining followingincubations in tumour (HT1080, closed symbols) and murine liver (opensymbols) homogenate for the indicated time. This is representative datafrom multiple experiments. Chromatographic separations were establishedto monitor the dephosphorylation of each molecule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the selection of aproteolytic cleavage site which allows for the selective release of avascular disrupting agent, such as colchicine, at the tumourvasculature.

According to a first aspect of the invention there is provided acompound, or pharmaceutically acceptable salt thereof, comprising avascular disrupting agent (VDA) associated with a MMP proteolyticcleavage site. The term “associated with” in the context of theinvention is intended to include all direct and indirect means ofassociation, generally covalent, including, but not limited to, chemicalcross-linking or peptide bond linkage.

Compounds of the invention may be in the form of salts. In particular,the salts may be pharmaceutically acceptable salts. The pharmaceuticallyacceptable salts of the present disclosure can be synthesized from theparent compound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred, Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, thedisclosure of which is hereby incorporated by reference; see also Stahlet al, Eds, “Handbook of Pharmaceutical Salts Properties Selection andUse”, Verlag Helvetica Chimica Acta and Wiley-VCH, 2002.

The disclosure thus includes pharmaceutically-acceptable salts of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. For example the conventional non-toxic saltsor the quaternary ammonium salts which are formed, e.g. from inorganicor organic acids or bases. Examples of such acid addition salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, tosylate, and undecanoate. Base salts includeammonium salts, alkali metal salts such as sodium and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine,lysine, and so forth. Also, the basic nitrogen-containing groups may bequaternized with such agents as lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethyl bromidesand others.

In a preferred aspect the invention provides a compound of formula (I)

X—Y  (I)

whereinX is a vascular disrupting agent (VDA);Y is a matrix metalloproteinase (MMP) proteolytic cleavage site.

Compounds according to the invention provide prodrugs which areconverted to an, active and potent VDA within the tumour environment byoverexpressed MMPs. Tumour selective activation of a prodrug of theinvention increases tumour levels, and decreases systemic levels, of theVDA and optionally additional active ingredients thereby greatlyincreasing their therapeutic index and efficacy.

VDAs comprise a multi-ring system, for example a fused or unfusedbicyclic or tricyclic ring system. Thus X includes any multi-ring systemof a VDA that is capable of binding to and disrupting tumour bloodvessels.

VDAs can be divided into three classes:

(i) those compounds that interact with tubulin at the colchicine bindingsite on tubulin;(ii) those compounds that share a common binding site on tubulin withthe Catharanthus (Vinca) alkaloids;(iii) compounds that promote the formation of stable microtubules in amanner similar to paclitaxel, a novel taxane diterpenoid isolated fromthe bark of the Pacific yew.

In a preferred aspect of the invention the VDA is a tubulin bindingagent. The tubulin binding agent may be selected from the groupconsisting of i) those which interact with tubulin at the colchicinebinding site: including but not limited to colchicine (includingcolchicine analogues such as N-acetylcolchinol-O-phosphate (ZD6126) andABT-751), colchicinoids, combretastatins phenstatin, podophyllotoxins,steganacins, amphethinile and stilbenes; and ii) those which interactwith the Vinoa binding site of tubulin: including but not limited tovincristine, vinblastine, vinflunine, maytansinoids, phomopson A,rhizoxin, auristatin (including analogues thereof) and dolastatin

In a preferred aspect of the invention the VDA is a tubulin bindingagent that interacts with the colchicine binding site within tubulin. Inone embodiment of the invention the VDA is colchicine or ananalogue/derivative thereof. Colchicine analogues or derivatives mayinclude, but are not limited to, azademethylcolchicine, azacolchicine,N-methyl desacetylcolchicine, desacetylcolchicine.

In an embodiment of the invention the VDA is a non-peptide VDA. Forexample a VDA according to the invention may be a tubulin binding agentthat is not auristatin or a derivative thereof.

Alternatively the VDA may be a tubulin binding agent selected from thegroup consisting of, but not limited to, combretastatins (e.g.combretastatin A-4 3-O-phosphate), auristatin (including analoguesthereof), dolastatin; and flavenoids (e.g. tumour necrosis factor a and5,6-dimethylxanthenone-4-acetic acid (DMXAA), flavone acetic acid(FAA)). Thus in an alternative embodiment of the invention the VDA is acombretastatin.

The invention includes any member of the MMP family. Proteolyticcleavage at the MMP cleavage site by an MMP releases the VDA, and anyother active agent associated with the MMP cleavage site, in activeform.

The MMP family is divided into eight structural groups: five of whichare secreted MMPs and three of which are membrane-type MMPs (MT-MMPs).MT-MMPs are localised on the cell surface. The invention includessecreted MMPs and membrane type MMPs.

In a preferred aspect of the invention the MMP is a membrane-type(MT-MMP). Thus the invention provides a compound of formula I wherein Ycomprises a peptide sequence that is selectively cleaved by a MT-MMP.The MT-MMP may be selected from the group consisting of

(i) type I transmembrane type MT-MMPs for example MMP-14 (MT1-MMP),MMP-15 (MT2-MMP), MMP-16 (MT3-MMP) and MMP-24 (MT5-MMP);(ii) glycosyl phosphatidylinositol (GPI)-anchored structural group ofMT-MMPs for example MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP);(iii) type II transmembrane class for example MMP-23.

The MMP cleavage site may comprise any peptide sequence having an amidebond, typically a peptide bond, which is cleavable by a MMP. PreferablyY is a peptide sequence comprising between two and twenty amino acids,for example between six and ten amino acids (e.g. 6, 7 or 8 aminoacids). The amino acids may be D-amino acids or L-amino acids.

The MMP proteolytic cleavage site may comprise the sequence:

(i) P3′-P2′-P1′-P1-P2-P3

wherein P1′ to P3′ and P1 to P3, which may be the same or different, areamino acid residues and wherein proteolytic cleavage takes place acrossthe bond between residues P1′ and P1.

Preferably MMP proteolytic cleavage site is MT-MMP specific cleavagesite such that cleavage between P1 and P1′ selectively takes place by aMT-MMP for example MT-MMP14.

The MMP proteolytic cleavage site may comprise the sequence:

(ii) P3′-P2′-P1′-P1-P2-P3-P4

wherein P1′ to P3′ and P1 to P4, which may be the same or different, areamino acid residues and wherein proteolytic cleavage takes place betweenresidues P1′ and P1.

The MMP proteolytic cleavage site may comprise the sequence:

(iii) P4′-P3′-P2′-P1′-P1-P2-P3-P4wherein P1′ to P4′ and P1 to P4, which may be the same or different, areamino acid residues and wherein proteolytic cleavage takes place betweenresidues P1′ and P1.

In a preferred aspect of the invention P1 and P1′ are different.

Preferably the MMP cleavage site comprises a peptide sequence which isselectively cleaved by a MT-MMP. Thus, in a preferred aspect theinvention provides a compound of formula (I) wherein Y comprises thesequence (i), (ii) or (iii) in which P1′ is a hydrophobic amino acid. Asused herein, the terms “hydrophobic” may refer to amino acids having ahydrophobicity that is greater than or equal to −1.10 and/or a netcharge that is greater than or equal to 0 as described in Fauchere andPliska Eur. J. Med Chem. 10:39 (1983). A hydrophobic or non-polar aminoacid may also refer to an amino acid having a side chain that isuncharged at physiological pH, is not polar and that is generallyrepelled by aqueous solution. A hydrophobic amino acid may be selectedfrom the group consisting of leucine, phenylalanine, proline, alanine,tryptophan, valine, isoleucine, methionine, tyrosine and threonine. P1′may be an amino acid with an aliphatic side chain. Alternatively P1′ maybe an amino acid with an aromatic side chain. P1′ may also includenon-natural hydrophobic amino acids for example homophenylalanine (Hof).

A preferred aspect of the invention provides a compound of formula (I)wherein Y comprises the sequence (i), (ii) or (iii) in which P1 is apolar amino acid for example an amino acid selected from the groupconsisting of Asparagine (N), Serine (S) or Glycine (G). To improveselectivity for a MT-MMP at the cleavage site, it is preferred that P1is not proline. Thus in an embodiment of the invention P1 is an aminoacid excluding proline. In a further embodiment of the invention, P1 isglycine.

A preferred aspect of the invention provides a compound of formula (I)wherein Y comprises the sequence (i), (ii) or (iii) in which P2′ isselected from the group consisting of polar, uncharged amino acidsand/or basic amino acids including for example Arginine (R), Alanine(A), Leucine (L), Aspartic Acid (D), Tyrosine (Y), Threonine (T), Serine(S) and Proline (P). To improve selectivity for a MT-MMP at the cleavagesite, P2′ may be a methylated amino acid for example N-methyl tyrosine.The present inventors have found that cleavage by non MT-MMPs, forexample MMP2, takes place at both the P1′-P2′ and P2′-P3′ sites when P2′is tyrosine. Thus in an embodiment of the invention P2′ is not tyrosine.

As used herein, the P3′ residue may comprise any amino acid. In oneembodiment of the invention P3′ is Leucine (L). Where present, the P4′residue may comprise an amino acid with a nucleophilic side chain forexample Lysine (L), Cysteine (C), Serine (S), Tyrosine (Y), Threonine(T), Glutamic acid (E), or Aspartic acid (D).

A preferred aspect of the invention provides a compound of formula (I)wherein Y comprises the sequence (i), (ii) or (iii) in which P2 isselected from the group consisting of acidic (for example glutamicacid), basic, hydrophobic or polar amino acids. P2 may also includenon-natural hydrophobic amino acids for example citrulline (Cit). Toimprove selectivity for a MT-MMP at the cleavage site, it is preferredthat P2 is not proline. Thus in an embodiment of the invention P2 is anamino acid excluding proline.

A preferred aspect of the invention provides a compound of formula (I)wherein Y comprises the sequence (i), (ii) or (iii) in which P3 is apolar amino acid for example selected from the group consisting ofGlycine (G), Alanine (A), Serine (S), Leucine (L), Isoleucine (I) andThreonine (T). It is preferred that P3 is not proline. Thus in anembodiment of the invention P3 is not proline. In a further embodimentof the invention P3 is serine.

A preferred aspect of the invention provides a compound of formula (I)wherein Y comprises the sequence (ii) or (iii) in which P4 is an aminoacid containing a substantial side chain, for example an amino acidhaving a basic group in its side chain, including amino acids selectedfrom the group consisting of Arginine (R) and Lysine (K). To improveselectivity for a MT-MMP at the cleavage site, it is preferred that P4is arginine. Thus in an embodiment of the invention P4 is arginine.

A preferred aspect of the invention provides a compound of formula (I)wherein Y comprises the sequence (i), (ii) or (iii) wherein none of theresidues are proline and wherein P4 is arginine.

Each of residues P1 to P3 or P4 may be different. Alternatively oradditionally each of residues P1′ to P3′ or P4′ is different.

One embodiment of the invention provides a compound of formula (I)wherein Y comprises the sequence (i), (ii) or (iii) in which P1 and/orP2 and/or P3 are an amino acid reside other than proline. Furthermore,in this embodiment P4 is arginine.

In a further embodiment of the invention there is provided a compound offormula (I) wherein Y comprises the sequence (i), (ii) or (iii) in whichP1′ is a hydrophobic amino acid and in which P1 and/or P2 and/or P3 arean amino acid reside other than proline.

In a yet further embodiment of the invention there is provided acompound of formula (I) wherein Y comprises the sequence (ii) or (iii)in which P1′ is a hydrophobic amino acid and P4 is arginine.Furthermore, in this embodiment P1 and/or P2 and/or P3 are an amino acidreside other than proline.

In another embodiment P1′ is homophenylalanine. In a yet further, orpreferred, embodiment, P1 is Gly.

The MMP proteolytic cleavage site, Y, may comprise the amino acidsequence (iv)

-Hof-Gly-  (iv)

The MMP proteolytic cleavage site, Y, may comprise the amino acidsequence (v)

-P2′-Hof-Gly-P2-  (v)

wherein P2 and P2′ are as defined herein.

The MMP proteolytic cleavage site, Y, may comprise the amino acidsequence (vi)

-P2′-Hof-Gly-P2-P3-P4-  (vi)

wherein P2′ to P4′ and P2 are as defined herein. Preferably P4 isarginine.

Preferably P3 is not proline.

In an embodiment the invention provides a compound of formula (I)wherein Y comprises the sequence—Leu-Tyr-Hof-Gly-Cit-Ser-Arg-. (SEQ IDNO: 1)

In a further embodiment the invention provides a compound of formula (I)wherein Y does not include the sequence—Leu-Gly-Leu-Pro- whereincleavage takes place between Leu-Gly residues.

The invention may provide a compound of formula (I) wherein the cleavagesite, Y, comprises a sequence of amino acids in which one or more aminoacids in the sequence are glycosylated to enhance hydrophilicity and assuch solubility. Glycosylation of residues may be as follows:

O-glycosylation of amino acids in the sequence through the side-chainsof serine, threonine, tyrosine;N-glycosylation of amino acids in the sequence through the side-chainsof aspartic acid, glutamic acid; and/orS-glycosylation of amino acids in the sequence through cysteine.

The invention may provide a compound of formula (I) wherein the cleavagesite, Y, comprises a sequence of amino acids in which one or more aminoacids in the sequence are phosphorylated to enhance hydrophilicity andas such solubility. Suitable amino acids include Serine, Threonine,Tyrosine. In an embodiment of the invention there is provided a compoundof formula (I) wherein Y comprises the sequence (i) (ii) or (iii) inwhich P2′, for example tyrosine, is phosphorylated.

In a further embodiment of the invention there is provided a compound offormula (I) wherein Y comprises the sequence (i) (ii) or (iii) in whichP3, and optionally P2′, is/are phosphorylated amino acids.

The invention also provides a compound of formula (I) wherein thecleavage site, Y, includes a peptide analogue, for example a peptidemimic, in which, by way of example an amide bond is replaced witholefinic bonds, Nα- and/or Cα-methylated amino acids, unnatural aminoacids and other approaches known in the art. Such peptidomimeticapproaches are used in the art to enhance the specificity of cleavagethereby serving to diminish undesired enzymatic hydrolysis. In anembodiment of the invention Y includes an analogue wherein one or moreamide bonds within the amino acid sequence are replaced with Nα- and/orCα-methylated amino acids.

An embodiment the invention comprises a compound of formula (I) whereinY comprises a C-terminal site and an N-terminal site and wherein saidC-terminal site is directly or indirectly linked to X and saidN-terminal site is directly or indirectly linked to a further moiety forexample c or Z as described hereinbelow.

An alternative embodiment of the invention comprises a compound offormula (I) wherein Y comprises a C-terminal site and an N-terminal siteand wherein said N-terminal site is directly or indirectly linked to Xand said C-terminal site is directly or indirectly linked to a furthermoiety for example c or Z as described hereinbelow.

In one preferred embodiment the invention there is provided a compoundof formula (I) wherein X is colchicine or an analogue thereof and Y is apeptide comprising the amino acid sequence (i), (ii), (iii), (iv), (v)or (vi) as defined herein.

In a preferred aspect the invention provides a compound of formula (II)

X—Y-c  (II)

wherein X and Y are as defined herein;c is an end group or “capping group”. Capping groups may be used to capa peptide chain in pharmaceutical use in order to prevent non-specificdegradation of the peptide for example by enzymes other than MMPs. c mayinclude any appropriate moiety on the N- or C-terminus acting as ablocking group selected from the group including, but not limited to,aliphatics, aromatics, polycyclics, carbohydrates (e.g. simple sugars),amino acids (including D-amino acids). To improve solubility, c may be ahydrophilic group for example any of the aforementioned bearingadditional polar functional groups (e.g. acids, amines, alcohols,phenols). c may be represented by the formula (c)_(n) wherein n is aninteger between 1 and 5. In an embodiment of the invention, c isrepresented (c)_(n) wherein c is an amino acid (e.g. a non-natural aminoacid) and n is 3 In an embodiment of the invention c is not serine wheren is 3. In a further embodiment c is not serine or quinic acid.

The present invention may further provide a “linker”. The linker may beprovided at the C and/or N terminus of Y. Preferably the linker isprovided at the C terminus of Y.

Preferably the linker is continuous with the amino acid sequence of Y.The linker may include any moiety that is associated with Y and whichmay be removed chemically, enzymatically or decompose spontaneously. Thelinker may consist of a single amino acid (e.g. tyrosine) or maycomprise an amino acid sequence. Where the linker comprises a sequenceof amino acids, the sequence may provide a hydrophilic region that mayfacilitate cleavage by an MMP at Y. O-glycosylation of suitable aminoacids in the sequence namely serine, threonine and tyrosine may enhancehydrophilicity and as such solubility.

Thus in a preferred aspect of the invention there is provided a compoundof formula (III)

X-a-Y  (III)

wherein X and Y are as defined herein; anda is a linker wherein the linker is directly or indirectly associatedwith X.

In an embodiment the invention provides a compound of formula (IV)

X-a-Y-c  (IV)

wherein X, a, Y and c are as defined herein.

In a yet further preferred aspect of the invention there is provided a“spacer” which may be the same as, or different to, the linker describedherein. The spacer may be provided at the C and/or N terminal of Y.Preferably the spacer is provided at the N terminus of Y and serves toprevent unwanted removal of c during synthesis. The spacer may bedirectly or indirectly associated with Y. The spacer may include anysingle amino acid (e.g. β-alanine), amino acid sequence, a succinylgroup. Thus the invention preferably provides a compound of formula (V)

X—Y-b-c  (V)

wherein X, Y, and c are as defined herein;b is a spacer as defined herein.

In a further embodiment the invention provides a compound of formula(VI)

X-a-Y-b-c  (VI)

wherein X, Y, a, b and c are as defined herein.

In one embodiment of the invention there is provided a compound offormula (VI) wherein X is colchicine (or an analogue thereof), Y is apeptide comprising the amino acid sequence (i), (ii), (iii), (iv), (v)or (vi) as defined herein, a is tyrosine and b is alanine.

In a second aspect of the invention there is provided a compound, orpharmaceutically acceptable salt thereof, of formula (VII)

X—Y-Z  (VII)

wherein X and Y are as defined herein; Z is an anti-cancer agent.

Preferably Z is an anticancer agent selected from the group consistingof a vascular disrupting agent, which may the same as or different to X,an antimetabolite (e.g. 5-fluorouracil), a cytotoxic oranti-proliferative agent (e.g. anthracycline (e.g doxorubicin), vincaalkaloid, taxane, cytotoxic nucleotide), a biotoxin, radiotherapeutic,hormonal agent or any natural products or agents known to induce acytotoxic, cytostatic, anti-angiogenic or vascular disrupting effect.

In a preferred aspect of the invention there is provided a compound offormula (VIII)

X-a-Y—Z  (VIII)

wherein X, a, Y and Z are as defined herein.

In a yet preferred aspect of the invention there is provided a compoundof formula (IX)

X-a-Y-b-Z  (IX)

wherein X, a, Y, b and Z are as defined herein. In this aspect of theinvention, the purpose of the spacer b is to convert the N-terminalamine of Y into a carboxylic acid to allow attachment of a compound Zwherein Z bears a free amine (for example where Z is doxorubicin). WhereZ bears a free carboxylic acid, b is not required.

In a preferred aspect of the invention there is provided a compound offormula (VII) wherein X is colchicine or a derivative thereof forexample azademethylcolchicine and Z is a cyctotoxic agent for exampledoxorubicin. Preferably still Y is a peptide comprising the amino acidsequence (i), (ii), (iii), (iv), (v) or (vi) as defined herein.

In a further preferred aspect of the invention there is provided acompound of formula (VII) wherein X and Z are selected from colchicineor an analogue or derivative thereof. Also provided is a compound of theinvention wherein X and Z may both be colchicine. In this aspect, Y maybe a peptide comprising the amino acid sequence (i), (ii), (iii), (iv),(v) or (vi) as defined herein.

Compounds according to the invention may be prepared by solid-phase, forexample attached to a polymeric support, or solution-phase synthesis forexample in the presence of a coupling agent or using a convergentsynthesis.

Thus a further aspect of the invention provides a process for preparinga compound according to the invention the process comprising the stepsof

i) providing a solid support attached to X;ii) optionally attaching a linker a to the C or N terminal of X;iii) attaching amino acid residues step-wise to the C or N terminal ofX, or the linker attached to X in (ii), to provide the peptide sequenceY containing the MMP proteolytic cleavage sequence;iii) optionally attaching a capping group c to the respective C or Nterminal of Y to provide a compound of formula (II) or (IV).

In a preferred process the solid support is any polymeric support suchas any polystyrene based or PEG based resin for example trityl-basedresins. The linker may be a succinate or malonate derivative for examplesuccinic anhydride.

A yet further aspect of the invention provides a process for preparing acompound according to the invention the process comprising the steps of

i) preparing peptide sequence Y;ii) attaching a capping group c to the respective C or N terminal of Y;iii) preparing a solution of X and the capped peptide prepared in (ii)in the presence of a coupling agent and isolating the desired compound.

In a preferred process Y is prepared by the step wise attachment ofamino acids to a solid support to provide a peptide sequence.Alternatively, sequence Y may be synthesised in solution.

Any coupling agent known in the art may be used in a process of theinvention for example EDAC, DCC, DiC, PyBOP, HCTU etc in a suitablesolvent (e.g. DMF, THE etc.

In an alternative solution phase synthesis, one or more amino acids ofthe C-terminus of sequence Y may be conjugated to X in solution, toallow the remainder of the peptide sequence (e.g. pre-synthesised on asolid support as described previously) to be conjugated in solution in aconvergent synthesis.

A preferred process of the invention provides for the attachment of X tothe C terminus of Y. Furthermore the process may include the attachmentof Z to the N-terminus of Y. For example the process may comprise thesteps of:

i) introduction of an amine group to X for attachment to the C terminusof Y; and optionallyii) introduction of a carboxylic acid group (or isothiocyanate orisocyanate group) to Z for attachment to the N terminus of Y.

A further preferred process provides for the attachment of X to theN-terminus of Y.

Furthermore the process may include the attachment of Z to theC-terminus of Y. For example the process may comprise the steps:

i) introduction of a carboxylic acid group (or isothiocyanate orisocyanate group) to X for attachment to the N terminus of Y; andoptionallyii) introduction of a amine group to Z for attachment to the C terminusof Y.

In a preferred process of the invention, X is colchicine or an analogueor derivative thereof e.g. azademthylcolchicine and Z is a cytotoxicagent e.g. doxorubicin.

In a further aspect the invention provides the use of a MMP proteolyticcleavage site, in particular an MT-MMP specific cleavage site such asdefined herein, in the site specific activation of a VDA. The term “sitespecific activation” as used herein means, in general terms and notlimited to, the activation of a VDA by site specific cleavage at the MMPproteolytic cleavage site. Site specific cleavage at the proteolyticcleavage site is expected to take place concomitantly with the releaseand hence activation of the VDA.

Pharmaceutical Compositions and Uses

other aspects the invention provides a compound, or pharmaceuticallyacceptable salt thereof, as hereinbefore described for use in medicine.In further aspects, there is provided a pharmaceutical formulationcomprising a compound as hereinbefore described. The formulation maycontain at least one additional pharmaceutically acceptable componente.g. an excipient, diluent or carrier. Preferably the formulation isintended for parenteral administration.

The invention provides a pharmaceutical formulation comprising acompound according to the invention. In a preferred embodiment, thecompound is of formula (VII).

In a preferred aspect of the invention said composition includes apharmaceutically acceptable carrier or diluent.

The compositions of the invention are typically administered ineffective amounts. An “effective amount” is that amount of a compositionthat alone, or together with further doses, produces the desiredresponse. When administered, the pharmaceutical compositions of thepresent invention are administered in pharmaceutically acceptablepreparations. Such preparations may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers and optionally other therapeutic agents (forexample, cisplatin; carboplatin; cyclosphosphamide; melphalan;carmusline; methotrexate; 5-fluorouracil; cytarabine; mercaptopurine;daunorubicin; doxorubicin; epirubicin; vinblastine; vincristine;dactinomycin; mitomycin C; taxol; L-asparaginase; G-CSF; etoposide;colchicine; derferoxamine mesylate; and camptothecin).

The compositions of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. In the case of treating a particular disease, such ascancer, the desired response is inhibiting the progression of thedisease. This may involve only slowing the progression of the diseasetemporarily, although more preferably, it involves halting theprogression of the disease permanently. This can be monitored by routinemethods known in the art.

Administration of the compositions to mammals other than humans, (e.g.for testing purposes or veterinary therapeutic purposes), is carried outunder substantially the same conditions as described above. A subject,as used herein, is a mammal, preferably a human, and including anon-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients.

Pharmaceutical compositions may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal. Compositions suitable for oral administration may bepresented as discrete units, such as capsules, tablets, lozenges, eachcontaining a predetermined amount of the active compound. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as syrup, elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of compound, whichis preferably isotonic with the blood of the recipient. This preparationmay be formulated according to known methods using suitable dispersingor wetting agents and suspending agents. The sterile injectablepreparation also may be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1, 3-butane diol. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution, and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono- or di-glycerides. Inaddition, fatty acids such as oleic acid may be used in the preparationof injectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

The compounds according to the invention may be used to treat a diseaseor condition associated with tissue over-expressing an MMP, inparticular a MT-MMP. Thus the invention provides a method to treatcancer in a subject comprising administering an effective amount of acompound according to the invention. In a preferred method of theinvention said subject is human.

As used herein, the term ‘cancer’ refers to cells possessing thecapacity for autonomous growth i.e., an abnormal state or conditioncharacterised by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathological type or stage of invasiveness. Theterm ‘cancer’ includes malignancies of epithelial, endodermal andmesenchymal origin, particularly carcinomas and sarcomas, such as thoseaffecting the respiratory system (mouth, nose, trachea, lung),gastrointestinal tract (tongue, esophagus, stomach, small intestines,colon, liver, pancreas, gall bladder, rectum), endocrine system(thyroid, pituitary, adrenal glands), genito-urinary tract (urinarybladder, kidney), reproductive system (breast, ovaries, uterus, cervix,prostate, penis, scrotum, testes), skin (melanocytes, epidermis,endodermis), nervous system (brain, spinal cord, glial cells, neurons)and lymphoid system.

The term ‘carcinoma’ is art recognised and refers to malignancies ofepithelial origin including respiratory system carcinomas,gastrointestinal system carcinomas, endocrine system carcinomas,genito-urinary tract carcinomas, skin carcinomas, and carcinomas of thereproductive system. The term “carcinoma” also includes“adenocarcinomas” referring to carcinomas deriving from glandulartissue, “squamous carcinomas” referring to carcinomas of squamousorigin, and “carcinosarcomas” referring to tumours composed ofcarcinomatous and sarcomatous tissue. Exemplary carcinomas include thoseforming from the epithelia of the cervix, prostate, breast, nose, headand neck, oral cavity, esophagus, stomach, liver, pancreas, colon,ovary, urinary bladder and lung, particularly non-small lung carcinoma.

The term ‘sarcoma’ is art recognised and refers to malignancies of softtissues or connective or supportive tissue, including bone, cartilage,adipose tissue, smooth muscle, skeletal muscle, nerve sheath, bloodvessels, mesothelium, and gastrointestinal stroma. Further types ofcancer include “leukaemias” which refer to tumours deriving from whiteblood cells, and “lymphomas” referring to tumours of the lymphoidsystem.

A pharmaceutical formulation comprising a compound according to theinvention may be administered in combination, either sequentially or ata substantially similar time, as an anti-cancer agent (chemotherapeuticagent) including, but not limited to, an antimetabolite (e.g.5-fluorouracil), a cytotoxic or anti-proliferative agent (e.g.anthracycline, vinca alkaloid, taxane, cytotoxic nucleotide), abiotoxin, radiotherapeutic, hormonal agent or any natural products oragents known to induce a cytotoxic, cytostatic, anti-angiogenic orvascular disrupting effect.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and may be performed either for prophylaxis or during thecourse of clinical pathology.

In a further aspect the invention provides the use of a compoundaccording to the invention in the manufacture of a medicament to treatcancer.

In one aspect of the present invention the compounds or compositions ofthe invention may be used to treat an inflammatory disorder and/or aninflammatory response. Thus, according to a further of the inventionthere is provided a method to treat an inflammatory disorder in asubject comprising administering an effective amount of a compoundaccording to the invention.

The inflammatory disorder may be selected from the group consisting ofconsisting of atherosclerosis, rheumatoid arthritis, osteoarthritis,gout, lupus erythematosus, scleroderma, Sjorgen's syndrome, poly- anddermatomyositis, vasculitis, tendonitis, synovitis, bacterialendocarditis, periodontitis, osteomyelitis, psoriasis, pneumonia,fibrosing alveolitis, chronic bronchitis, bronchiectasis, emphysema,silicosis, pneumoconiosis, tuberculosis, ulcerative colitis, Crohn'sdisease, chronic inflammatory demyelinating polyradiculoneuropathy,chronic inflammatory demyelinating polyneuropathy, multiple sclerosis,Guillan-Barre Syndrome and myasthemia gravis, mastitis, laminitis,laryngitis, chronic cholecystitis, Hashimoto's thyroiditis, andinflammatory breast disease. In one embodiment, the inflammatorydisorder may be the result of tissue or organ rejection aftertransplantation. In particular embodiments the inflammatory disorder isselected from the group consisting of atherosclerosis, rheumatoidarthritis, osteoarthritis, sepsis and polyarthritis.

The compounds of the invention may be used to treat heart failure. Alsoprovided is a use of a compound as described herein for the manufactureof a medicament to treat heart failure.

In one embodiment of the present invention the compounds of theinvention may be useful in treating a wound (e.g. ulcers, lesionsincluding cutaneuous cuts or burns). Thus the invention provides amethod to treat a wound in a subject comprising administering aneffective amount of a compound according to the invention. In apreferred method of the invention said subject is human.

The compounds of the invention may also be used to treat conditions anddisorders associated with menstruation.

There is further provided a package or kit of parts comprising:

(1) a compound or composition described herein; together with(2) instructions to use the compound in a method or use describedherein.

The package defined herein may comprise more than one dosage unit inorder to provide for repeat dosing. If more than one dosage unit ispresent, such units may be the same, or may be different in terms of thedose of the compound composition and/or physical form.

The extent of protection includes counterfeit or fraudulent productswhich contain or purport to contain a compound of the inventionirrespective of whether they do in fact contain such a compound andirrespective of whether any such compound is contained in atherapeutically effective amount.

Included in the scope of protection are packages which include adescription or instructions which indicate that the package contains aspecies or pharmaceutical formulation of the invention and a productwhich is or comprises, or purports to be or comprise, such a formulationor species. Such packages may be, but are not necessarily, counterfeitor fraudulent.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

Materials and Methods Example 1 Synthesis of Immobilised ColchicineDerivative, Azademethylcolchicine

Synthesis of 1:

Ammonia solution (35%, 15 mL) was added to colchicine (750 mg, 1.88mmol, 1.00 eq) and the reaction mixture stirred at room temperatureovernight. The crude product was washed with KHSO₄ (1M, aq), dried withMgSO₄, filtered and concentrated under reduced pressure. It wassubsequently purified by flash chromatography on silica gel (gradientelution: CH₂Cl₂/methanol 95:5 to 10:1) to give 1 as a yellow solid (427mg, 1.11 mmol, 59%).

δ_(H) (600 MHz, CDCl₃), 7.99 (1H, broad s, NH), 7.56 (1H, d, J 2.1,C8-H), 7.32 (1H, d, J 10.7, C11-H), 6.88 (1H, d, J 11.0, C10-H), 6.52(1H, s, C4-H), 6.03 (2H, broad s, NH₂), 4.68 (1H, ddd, J 12.6, 6.5 and6.5, C7-H), 3.93 (3H, s, OCH₃), 3.88 (3H, s, OCH₃), 3.60 (3H, s, OCH₃),2.47 (1H, dd, J 13.4 and 6.2, C5-CH₂), 2.35 (1H, ddd, J 13.4, 12.7 and6.9, C5-CH₂), 2.29-2.23 (1H, m, C6-CH₂), 1.98 (3H, s, CH₃), 1.90-1.88(1H, m, C6-CH₂); ES m/z (%) 385 [M⁺+H](100).

Synthesis of 2:

HCTU (642 mg, 1.55 mmol, 1.50 eq) and diisopropylethylamine (DiPEA, 516μL, 404 mg, 3.11 mmol, 3.00 eq) were added to a solution ofFmoc-tyr(tBu)-OH (714 mg, 1.55 mmol, 1.50 eq) in DMF (10 mL). Afterstirring at room temperature for 5 minutes, 1 (398 mg, 1.04 mmol, 1.00eq) was added to the solution. The reaction mixture was stirred at 50°C. for 22 h. DMF was removed in vacuo and the resultant oil wasdissolved in CH₂Cl₂ (20 mL). The organic phase was washed with KHSO₄(aq, 2×20 mL), dried with MgSO₄ and concentrated under reduced pressure.The crude product was purified by flash chromatography (gradientelution: CH₂Cl₂/methanol 100:0 to 99:1 to 98:2) to give 2 as a yellowsolid (530 mg, 642 μmol, 67%).

δ_(H) (600 MHz, CDCl₃), 10.42 (1H, broad s, NH), 9.02 (1H, d J 10.7,C11-H), 7.75 (2H, d, J 7.2, C23-H, C24-H), 7.54 (2H, d, J 7.2, C20-H,C27-H), 7.45 (1H, d, J 11.0, C10-H), 7.39 (2H, dd, J 7.2 and 7.2, C22-H,C25-H), 7.29 (2H, dd, J 6.6 and 6.6, C21-H, C26-H), 7.19 (1H, broad s,C8-H), 7.03 (2H, d, J 7.9, C14-H, C17-H), 6.81 (2H, d, J 7.9, C15-H,C16-H), 6.50 (1H, s, C4-H), 5.88 (1H, broad s, NH), 5.25 (1H, broad s,C12-H), 4.73-4.67 (1H, m, C7-H), 4.43 (1H, dd, J 10.0 and 7.6, C18-CH₂),4.28 (1H, dd, J 10.0 and 7.2, C18-CH₂), 4.16 (1H, dd, J 7.2 and 6.19,C19-H), 3.93 (3H, s, OCH₃), 3.88 (3H, s, OCH₃), 3.62 (3H, s, OCH₃), 3.21(1H, dd, J 13.1 and 4.8, C13-CH₂), 3.11 (1H, dd, J 13.1 and 5.5,C13-CH₂), 2.53 (1H, dd, J 13.4 and 6.2, C5-CH₂), 2.40 (1H, ddd, J 13.4,12.7 and 6.9, C5-CH₂), 2.22-2.15 (1H, m, C6-CH₂), 1.88 (3H, s, CH₃),1.80 (1H, ddd, J 11.5, 11.3 and 6.9, C6-CH₂), 1.22 (9H, s, C(CH₃)₃); ESm/z (%) 826 [M⁺](100).

Synthesis of 3:

TFA (2 mL) was added to a solution of 2 (486 mg, 589 μmol, 1.00 eq) andthe reaction mixture stirred for 20 min. TLC indicated quantitativeconversion to the product. The product was concentrated under reducedpressure, with toluene co-evaporation to give 3 in quantitative yield.

δ_(H) (600 MHz, CDCl₃), 10.08 (1H, broad s, NH), 8.99 (1H, d J 10.7,C11-H), 7.71 (2H, d, J 6.2, C23-H, C24-H), 7.55 (1H, s, C8-H), 7.49 (2H,dd, J 6.5 and 6.5, C20-H, C27-H), 7.41 (1H, d, J 10.2, C10-H), 7.33 (2H,dd, J 6.2 and 6.2, C22-H, C25-H), 7.26-7.21 (2H, m, C21-H, C26-H), 6.91(2H, d, J 8.3, C14-H, C17-H), 6.56 (2H, d, J 7.2, C15-H, C16-H), 6.45(1H, s, C4-H), 5.93 (1H, broad s, NH), 5.28 (1H, s, NH), 4.95-4.90 (1H,m, C12-H), 4.60 (1H, ddd, J 11.7, 5.8 and 6.9, C7-H), 4.39 (1H, dd, J8.9 and 8.6, C18-CH₂), 4.29-4.24 (1H, m, C18-CH₂), 4.12 (1H, dd, J 6.9and 6.9, C19-H), 3.90 (3H, s, OCH₃), 3.84 (3H, s, OCH₃), 3.54 (3H, s,OCH₃), 3.08 (2H, d, J 5.2, C13-CH₂), 2.44 (1H, dd, J 13.4 and 6.2,C5-CH₂), 2.33-2.26 (1H, m, C5-CH₂), 2.15-2.09 (1H, m, C6-CH₂), 1.82 (3H,s, CH₃), 1.75-1.69 (1H, m, C6-CH₂); ES m/z (%) 770 [M⁺](100).

Preparation of 4:

2-Chlorotrityl chloride resin (Novabiochem, 100-200 mesh, substitution1.4 mmolg¹, 589 mg, 0.765 mmol, 1.00 eq) was suspended in a solution of3 (589 mg, 0.765 mmol, 1.00 eq), dimethylaminopyridine (10 mg, 76.5μmol, 0.01 eq), DiPEA (247 mg, 1.913 mmol, 333 μL, 2.50 eq) and pyridine(241 mg, 3.061 mmol, 248 μL, 4.00 eq) in THF (10 mL) and stirred for 6hours at 50° C. The resin was subsequently filtered and washedthoroughly with THF. The resin was then capped by washing the resincarefully with methanol (CH₂Cl₂:MeOH:DiPEA 17:2:1, 100 mL). Resin 4 wasdried overnight over P₂O₅. Dry resin weight: 593 mg (loading 56%).

General Procedure for Synthesis of Endopeptidase-Activated Pro-Drugs

As an example, peptide conjugate 5 was synthesised using conventionalsolid phase peptide synthesis, from immobilised colchicine derivative 4,using an Fmoc-based strategy.

Na-Fmoc strategy synthesis was achieved manually using2-hlorotritylderivatised resin. The resin was swelled thoroughly in DMF,followed by removal of the NFmoc protecting group by treatment with 20%v/v piperidine in DMF (3×3 min). All couplings were performed in DMF,employing 2.5-fold molar excesses of Na-Fmoc protected amino acids (withappropriate side-chain protecting groups), and activated usingHCTU/HOBt/DiPEA. Na-Fmoc deprotections were performed using 20%piperidine in DMF (3×3 min). The success of couplings and deprotectionswas monitored using the ninhydrinbased Kaiser test. Unsuccessfulcouplings were repeated. After the final Nα-Fmoc deprotection, thepeptide chain was endcapped with fluorescein isothiocyanate (2.50 eq, inthe presence of DiPEA, 1.50 eq). The success of this reaction was alsomonitored by the Kaiser test.

An additional β-alanine residue was incorporated into the sequence toovercome incompatibility of the thiourea linkage and the acidicconditions of cleavage (the thiourea can rearrange, and the carbonylcarbon of the preceding amide bond can undergo nucleophilic attack bythe sulphydryl-like function so formed. This leads to cleavage of theamide bond, with concomitant formation of a cyclic thiazolinone. Thethiazolinone can undergo rearrangement in the presence of aqueous acidto form a thiohydantoin).

On completion of the sequence, the resin was washed (DMF, CH2Cl2,CH2Cl2/MeOH) and dried in vacuo over KOH to constant weight. Peptideswere cleaved from the resin by mild acidolysis usingTFA-H2O-triisopropylsilane 95:2.5:2.5 for 2 h at RT, with simultaneoussidechain de-protection. Following cleavage, the TFA was removed underreduced pressure. The crude product was extracted into 95% aqueousacetic acid and lyophilised. Crude peptide was subsequently analysedusing reversed phase HPLC and purified using preparative HPLC(purity>97%). Pure fractions were combined and lyophilised. A C18preparative column (Zorbax Eclipse, 21.2×150 mm XDB-C18, Agilent) wasused for peptide conjugate purification. Mobile phases were as follows;Mobile Phase A: HPLC grade water and 0.045% TFA. Mobile Phase Bconsisted of 10% HPLC grade water, 90% acetonitrile and 0.045% TFA.Mobile phase was degassed by vacuum filtration through a 0.45 μm porecellulose nitrate filter (Sartolon, Sartorius, UK). Chromatography wasperformed at room temperature and the flow rate maintained at 21.2ml/min. Mobile phase gradients were optimised and scaled up from agradient previously designed in-house for structurally similarcompounds. UV absorbance detection was optimal at 255 nm. ES m/z (%)951.8 [M+H]2+(100). RP-HPLC (Gradient: 0 min 30% B; 5 min 35% B; 25 min80% B; 26 min 100% B; 27 min 100% B; 30 min 30% B) Rt=11.98 min.

Attachment of Colchicine to a Peptide Sequence Through its B-Ring to thePeptide N-Terminus

To enable attachment of a colchicine moiety through the peptideN-terminus, the following strategy will be used. The B-ring amine can bede-masked using published methods. Acylation with aspartic acid willintroduce a carboxylic acid to the molecule (from the amino acid sidechain) thereby enabling conjugation to the free amine at the

peptide N-terminus (see below).

Acetylation of the amide bond will also be examined, to assess whetherparent colchicine is released following MMP activation and subsequentexopeptidase degradation.

Attachment of Colchicine Analogue to a Peptide Sequence Through itsC-Ring to the Peptide N-Terminus

To enable attachment of a colchicine analogue through its N-terminus,the following strategy will be used. Acylation of azademethylcolchicinecan be effected as described previously. On de-masking of theBoc-protecting group, acylation with succinic anhydride (or amono-protected succinate or malonate (or similar) derivative) follows.Deprotection will then afford a carboxylic acid functionality suitablefor attachment to the peptide N-terminus.

Strategies for the Attachment of Doxorubicin to a Peptide Sequence:

N-Terminal Doxorubicin Linkage

To enable attachment of doxorubicin through the peptide N-terminus(following peptide synthesis using the immobilised colchicinederivatised resin previously described) it must first be modified tointroduce a carboxylic acid. Examples include reaction with succinicanhydride (strategy 1, below). However, by utilising the side chain ofaspartic acid (both natural amino acids), as shown below (strategy 2) anatural amino acid (as opposed to a foreign chemical entity) is releasedon metabolism:

Strategy 1: (Succinyl Spacer)

Strategy 2: (Amino Acid Spacer)

Strategy 3

In a manner analogous to that described in Strategies 1 and 2 thedoxorubicin sugar amine can be acylated by an amino acid, which onde-protection can be further derivatised by a succinate (or otherwise)spacer to yield a derivative which can be attached to the peptideN-terminus.

2) C-Terminal Doxorubicin Linkage

Protection of the amine group of doxorubicin with Dde (a commonly usedprotecting group in peptide chemistry and by our group) would allowimmobilisation of the agent onto a trityl-based (or otherwise) resin.Subsequent removal of the Dde group would de-mask the amine, allowing apeptide sequence to be constructed from this point (i.e. through theC-terminus). Standard Fmoc-based solid phase synthesis would produce apeptide sequence. An appropriately derivatised VDA could then beconjugated through the N-terminus. Resin cleavage and purification wouldbe as previously described.

Incorporation of Glycosylated Amino Acids

Amino acids with appropriate side chain functionality (e.g. serine,tyrosine, threonine) can be glycosylated (with mono-, di- ortrisaccharides) to produce peptides with enhanced aqueous solubility.Such a carbohydrate-derivatised moiety could be used in place of serine,for example (see scheme below).

Results

1) MMP-Activated Prodrug

-   -   pan MMP targeted

Structure:

(Hof=homophenylalanine; Cit=citrulline; VDA-2=azademethylcolchicine)

-   a) Prodrug-1 has been screened using normal mouse plasma, normal    mouse liver homogenate and experimental human tumour model    homogenate (HT1080 xenograft; known to express majority of MMPs) ex    vivo. Prodrug cleavage and metabolism were detected using LC/MS/MS.    -   a. Prodrug-1 was stable in plasma and liver, supporting systemic        stability of these therapeutics (FIG. 1).    -   b. Prodrug-1 was metabolised in tumour homogenate, supporting        activation of these therapeutics in tumours expressing MMPs        (FIG. 1).-   b) Prodrug-1 is cleaved rapidly at the Glycine-Homophenylalanine    (Gly-Hof) by recombinant MMP-2, MMP-9, MMP-10 and MMP-14 at least.    Demonstrated by LC/MS/MS and mass spectrometry (data not shown)-   c) Prodrug-1 was administered in vivo via the intraperitoneal route    to mice bearing subcutaneous HT1080 tumour model (expression of    majority of MMPs). Plasma, tissues and tumours were collected at    regular intervals post-dosing. Levels of prodrug and VDA2 were    assessed by LC/MS/MS.    -   a. Prodrug-1 accumulated and was detected in all tissues        analysed (FIG. 2).    -   b. Highest prodrug-1 levels were observed in the tumour (FIG.        2).    -   c. Prodrug-1 not detectable after 24 hours post-dosing. (FIG. 2)    -   d. VDA2 was detectable at low levels in normal tissues following        prodrug-1 administration (FIG. 3)    -   e. VDA2 levels were detected at high levels in tumour tissue        following prodrug-1 administration (FIG. 3)    -   f. VDA2 was still detectable at high levels in tumour and was        undetectable in normal tissues after 24 hours post-dosing with        prodrug-1 (FIG. 3)

2) MMP-Activated Prodrug (ICT-2588)

-   -   targeted to Membrane-type MMPs (MT-MMPs)

Structure:

-   d) Compound 1 was modified in order to change the MMP-targeting of    the compound from being pan-MMP to MT-MMP selective (Prodrug-2)    -   a. Arginine was incorporated in place of the Glutamic acid at        the P4 position    -   b. Proline was removed and replaced with Serine at the P3        position-   e) Prodrug-2 has been screened using normal mouse plasma, normal    mouse liver homogenate and experimental human tumour model    homogenates demonstrating high MT1-MMP (MMP-14) expression and    activity (HT1080) and low MT1-MMP expression and activity (MCF-7) ex    vivo. Prodrug-2 cleavage and metabolism were detected using    LC/MS/MS.    -   a. Prodrug-2 remained intact in plasma supporting systemic        stability of this therapeutic.    -   b. Prodrug-2 remained stable in murine liver homogenates    -   c. Prodrug-2 was metabolised rapidly in tumour homogenate        expressing high MT-MMP levels (HT1080) relative to tumour        homogenate expressing low MT-MMP levels (MCF7) (FIG. 4).    -   d. These data support the systemic stability of this prod rug        and the selectivity of activation by MT-MMPs.-   f) Prodrug-2 is cleaved differentially by MMPs as shown by LC/MS/MS    and mass spectrometry (data not shown):    -   a. Cleaved rapidly at the Glycine-Homophenylalanine (Gly-Hof) by        recombinant MMP-14.    -   b. Cleaved slowly at the Homophenylalanine-Tyrosine (Hof-Tyr) by        recombinant MMP-2. Demonstrating different cleavage to that        observed with prodrug-1.    -   c. Prodrug-2 is not cleaved by recombinant MMP-9, in contrast to        prodrug-1    -   d. These data support the MMP selective cleavage of prodrug-2-   g) Prodrug-2 was administered in vivo via the intraperitoneal route    to mice bearing subcutaneous HT1080 tumour model (MT1-MMP positive).    Plasma, tissues and tumours were collected at regular intervals    post-dosing. Levels of prodrug-2 and VDA2 were assessed by LC/MS/MS.    -   a. Prodrug-2 accumulated and was detected in all tissues        analysed (FIG. 5).    -   b. Highest prodrug-2 levels were observed in the tumour (FIG.        5).    -   c. Liver was representative of all normal tissues analysed.        (FIG. 5)    -   d. VDA2 was undetectable in plasma following administration of        prodrug-2 (FIG. 6)    -   e. High concentrations of VDA2 were detected in tumour following        prodrug-2 administration (FIG. 6)    -   f. Levels of VDA2 in tumour were 10 times higher than that        detected in normal tissues following administration of prodrug-2        (FIG. 6)    -   g. High levels of prodrug-2 and VDA2 were still detectable in        tumour 48 hours post administration.

3. Synthesised conjugates (based on MT-MMP prodrug) which have provedunsuccessful. Prodrugs including the following endcaps wereinsufficiently stable in plasma and/or liver

-   -   (1) 3×D-Serine    -   (2) quinic acid+2×D-Serine

Example 2

Dose Response Antitumour Study

Materials and Methods

Synthesis of ICT-2588—see Example 1

Synthesis of ICT-2552: Ammonia solution (35%, 15 mL) was added tocolchicine (750 mg, 1.88 mmol, 1.00 eq) and the reaction mixture stirredat room temperature overnight. The crude product was washed with KHSO4(1M, aq), dried with MgSO4, filtered and concentrated under reducedpressure. It was subsequently purified by flash chromatography on silicagel (gradient elution: CH2Cl2/methanol 95:5 to 10:1) to give ICT-2552 asa yellow solid (427 mg,

1.11 mmol, 59%). δH (600 MHz, CDCl3), 7.99 (1H, broad s, NH), 7.56 (1H,d, J 2.1, C8-H), 7.32 (1H, d, J 10.7, 011-H), 6.88 (1H, d, J 11.0,C10-H), 6.52 (1H, s, C4-H), 6.03 (2H, broad s, NH2), 4.68 (1H, ddd, J12.6, 6.5 and 6.5, C7-H), 3.93 (3H, s, OCH3), 3.88 (3H, s, 00H3), 3.60(3H, s, OCH3), 2.47 (1H, dd, J 13.4 and 6.2, C5-CH2), 2.35 (1H, ddd, J13.4, 12.7 and 6.9, C5-CH2), 2.29-2.23 (1H, m, C6-CH2), 1.98 (3H, s,CH3), 1.90-1.88 (1H, m, C6-CH2); ES m/z (%) 385 [M+1-1]+(100).

ICT-2552 and ICT-2588 (‘prodrug’) were administered as a single dose viathe intraperitoneal route to Balb/C mice bearing subcutaneous HT1080xenografts. Each of the nine dosing groups comprised 8 mice. Theanti-tumour effects of the compounds were assessed by determination oftumour volume and the existence of off-target toxicity was ascertainedvia monitoring mouse body weight.

The dosing groups evaluated ICT-2588 (prodrug) relative to the molarequivalent dose of ICT-2552 (warhead), and were:

-   -   10% DMSO/oil (solvent control)    -   ICT-2588 at 37.5 mg/kg; 50.0 mg/kg; 62.5 mg/kg; 75.0 mg/kg    -   ICT-2552 at 7.5 mg/kg; 10.0 mg/kg; 12.5 mg/kg; 15 mg/kg

Results

No significant loss in body weight was observed throughout the study(FIG. 7)

The weight of all mice was within the tolerated loss of 15% ofbodyweight, therefore the compounds are not classified as toxic to thewhole body system.

Example 3

Tumour Response Study

Results

The results of the study are shown in FIGS. 8 to 10 and in the followingTables. Tumour responses were observed at all tested dose levels ofICT-2522 and ICT-2588.

TABLE 1 Antitumour efficacy of compounds relative to untreated controlgroup Mean Median Maximum time to time Growth % weight Com- Dose RTV2RTV2 delay Signifi- loss pound (mg/kg) (days) (days) (days) cance ^(a)(day)^(b) Control — 2.3 2.2 — — 5 (6) ICT-2588 37.5 4.4 4.2 2.0 p < 0.013 (3) ICT-2588 50.0 5.5 6.0 3.8 p < 0.01 5 (3) ICT-2588  62.5^(c) 7.77.8 5.6 p < 0.01 4 (3) ICT-2588 75.0 6.8 6.6 4.4 p < 0.01 5 (3) ICT-2552 7.5 4.0 3.8 1.6 p < 0.01 3 (4) ICT-2552 10.0 4.4 4.2 2.0 p < 0.01 6 (6)ICT-2552 12.5 4.5 4.7 2.5 p < 0.01 6 (1) ICT-2552 15.0 4.1 4.1 1.9 p <0.01 5 (1) ^(a) Statistics worked out based on the time it takes thetumour to double in size from day 0. ^(b)Maximum weight loss well withinthe accepted levels of 15%. ^(c)One animal showed complete tumourremission (final measurement day 21)

A significant delay in tumour growth was observed at all evaluated dosesof ICT-2552 (warhead) and ICT-2588 (prodrug).

A relationship between dose of compound and degree of tumour growthdelay was observed with both compounds. The effects being greater withICT-2588 (prodrug)

One animal demonstrated complete tumour remission with ICT-2588 at adose of 62.5 mg/kg

TABLE 2 Comparison of antitumour efficacy between dose of ICT-2588(prodrug) and ICT-2552 (warhead) administered at the molar equivalentdose Growth Growth Significance of differen- Dose of Dose of delay withdelay with tial between equimolar ICT-2588 ICT-2552 prodrug warheaddoses of ICT-2588 (mg/kg) (mg/kg) (Days) (Days) and ICT-2552^(a) 37.57.5 2.0 1.6 p > 0.05 50.0 10.0 3.8 2.0 p < 0.05 62.5^(c) 12.5 5.6 2.5 p< 0.05 75.0 15.0 4.4 1.9 p < 0.01 ^(a)Statistics worked out based on thetime it takes the tumour to double in size from day 0. ^(c)One animalshowed complete tumour remission (final measurement day 21) ICT-2588(prodrug) induced a significantly greater antitumour response thanICT-2522 (warhead) when administered at equimolar doses

Example 4

Modifications to ICT-2588

i) Improvement of Prodrug Solubility Through Introduction ofPhosphorylated Amino Acid Side Chains

To increase prodrug solubility, phosphorylated amino acid side areintroduced chains into the peptide sequence of ICT-2588 (hydrolysable byplasma phosphatases). Phosphate-derivatised amino acids are commerciallyavailable, as benzyl-protected moieties, and are incorporated intopeptides using our synthetic methodology. We are synthesising andevaluating phosphorylation modification of two sites within the peptidesequence; P2′ (Tyr), P3 (Ser), and both P2′ and P3 (see FIG. 11). Inaddition, we are addressing the effect of phosphorylation of P2′ (Tyr)upon prodrug activation by MT-MMPs (cleave at P1-P1′) and MMP-2 (cleaveat P1′-P2′) to address the potential for further increasing MMPselectivity.

ii) Improvement of Solubility Via Modification of Prodrug EndcappingGroup

We are exploring the replacement of the fluorescein group with a morehydrophillic group (see FIG. 11) as alternative prodrugs with improvedsolubility. Our objective is to improve prodrug solubility whilstretaining MT-MMP selectivity, tumour selective activation and antitumouractivity. Prodrugs incorporating these alternative endcaps to ICT2588are being assessed in terms of compound solubility relative to parentprodrug in vitro, prodrug stability and activation in the HT1080 tumourmodel, mouse plasma and liver homogenate ex vivo, and retention andpotential improvement of MT-MMP selectivity.

iii) Modification of P2′ Site within Prodrug

The effects of modification of the P2′ site (Tyr) of the prodrug withreference to tissue stability, tumour cleavage and MMP activation exvivo are being assessed. The focus is on the P2′ site of the prodrug aswe have demonstrated cleavage by other non-MT-MMPs at both the P1′-P2′and P2′-P3′ sites. We are using prodrug variants incorporating the maintypes of amino acid side chain (Ala, Asp, Asn, Leu, Ser, Pro).

iv) Development of Dual-Headed Prodrug Incorporating Doxorubicin

Following treatment with ICT2588 a thin viable rim of tumour cells wasobserved at the tumour periphery. Current understanding of tumourvascularisation indicates that the viable rim is maintained by thevasculature of the immediately surrounding normal tissues. A consequenceof this is that administration of a VDA-releasing prodrug in combinationwith standard chemotherapy should increase further the antitumour effectobserved. We are developing a dual-headed prodrug containing ourazademethylcolchicine warhead (ICT2552) and a doxorubicin warhead(replacing the endcap) (see below). We are evaluating this dual-headedprodrug in terms of prodrug stability and activation in the HT1080tumour model, mouse plasma and liver homogenate ex vivo.

Derivatisation at the amine is required since attachment to the peptidewill be through the N-terminus. Current strategy is to acylate withsuccinic anhydride, yielding a succinate derivative bearing a freecarboxylic acid which can be linked to the peptide via an amide bond.Alternative strategy to be evaluated is to use the side chain ofaspartic acid with alkyl/aryl blocking group R.

Table 3 is a summary of stability data for a series of molecules withchanges in the P2′ position. Half lives are the mean taken from fourindependent experiments. Liver represent murine liver homogenate andtumour represents HT1080

Liver Tumour P2′ AA t½ (min) t½ (min) ICT2588 Ty 149 33 ICT3055 Asp 67 7ICT3053 Al 76 17 ICT3054 Ser 66 9 ICT3078 Asn 76 11 ICT3097 Pro 110 24ICT3080 Leu 134 48

1.-14. (canceled)
 15. A method of treating a solid tumor in a subjectcomprising administering an effective amount of a compound, orpharmaceutically acceptable salt thereof, comprising a vasculardisrupting agent (VDA) associated with a matrix metalloproteinase (MMP)proteolytic cleavage site, wherein the VDA is selected from the groupconsisting of azademethylcolchicine, colchicine, azacolchicine, N-methyldesacetylcolchicine, desacetylcolchicine, N-acetylcolchinol-O-phosphate,colchicinoids, combrestatins, phenstatin, podophyllotoxins, steganacins,amphethinile, stilbenes, flavonoids, vincristine, vinblastine,vinflunine, maytansinoids, phomopson A, rhizoxin, auristatin, anddolstatin, and the MMP proteolytic cleavage site comprises the aminoacid sequence -Leu-P2′-Hof-Gly-Cit-Ser-Arg-, wherein P2′ is an aminoacid selected from Asp, Ala, Ser, Asn, Pro, Leu, Arg and Thr.
 16. Themethod according to claim 15, wherein the VDA is selected from the groupconsisting of azademethylcolchicine, colchicine, azacolchicine, N-methyldesacetylcolchicine, and desacetylcolchicine.
 17. The method accordingto claim 15, wherein the compound further comprises a capping group c onthe N- or C-terminus of the peptide which prevents non-specificdegradation of the peptide by enzymes other than MMPs.
 18. The method ofclaim 15, wherein the amino acid at P2′ is methylated.
 19. The method ofclaim 16, wherein the VDA is azademethylcolchicine.
 20. The method ofclaim 15, wherein an anti-cancer agent is further linked to the peptidecomprising the MMP proteolytic cleavage site.
 21. The method of claim20, wherein the anti-cancer agent is selected from the group consistingof 5-fluorouracil, anthracycline, doxorubicin, vinca alkaloid, taxane, acytotoxic nucleotide, a biotoxin, radiotherapeutic, hormonal agent,colchicine, azademethylcolchicine, N-methyl desacetylcolchicine anddesacetylcolchicine.
 22. The method of claim 21, wherein the anti-canceragent is doxorubicin.
 23. The method of claim 15, wherein the compoundis in the form of a pharmaceutical formulation comprising the compoundand at least one additional pharmaceutically acceptable excipient,diluent or carrier.
 24. The method of claim 23, wherein thepharmaceutical formulation further comprises a second therapeutic agentselected from the group consisting of cisplatin, carboplatin,cyclophosphamide, melphalan, carmustine, methotrexate, 5-fluorouracil,cytarabine, mercatopurine, daunorubicin, doxorubicin, epirubicin,vinblastine, vincristine, dactinomycin, mitomycin C, taxol,L-asparaginase, granulocyte colony stimulating factor (G-CSF),etoposide, colchicine, deferoxamine mesylate and camptothecin.
 25. Themethod of claim 15, wherein the compound is of formula (I)X—Y  (I), wherein X is the VDA; and Y is the peptide comprising the MMPproteolytic cleavage site.
 26. The method of claim 25, wherein Y is apeptide sequence of between seven and ten amino acids.
 27. The method ofclaim 15, wherein the compound is of formula (II)X—Y-c  (II), wherein X is the VDA; Y is the peptide comprising the MMPproteolytic cleavage site; and c is a capping group which preventsnon-specific degradation of the peptide by enzymes other than MMPs. 28.The method of claim 27, wherein c is selected from the group consistingof fluorescein isothiocyanate and fluorescein.
 29. The method of claim27, wherein c is the formula (c)_(n) and wherein n is an integer between1 and
 5. 30. The method of claim 29, wherein c is a non-natural aminoacid and n is
 3. 31. The method of claim 15, wherein the compound is offormula (III)X-a-Y  (III), wherein X is the VDA; Y is the peptide comprising the MMPproteolytic cleavage site; and a is a linker directly or indirectlyassociated with X and wherein the linker is a single amino acid or aminoacid sequence.
 32. The method of claim 15, wherein the compound is offormula (IV)X-a-Y-c  (IV), wherein X is the VDA; Y is the peptide comprising the MMPproteolytic cleavage site; a is a linker directly or indirectlyassociated with X and wherein the linker is a single amino acid or aminoacid sequence; and c is a capping group which prevents non-specificdegradation of the peptide by enzymes other than MMPs.
 33. The method ofclaim 15, wherein the compound is of formula (V)X—Y-b-c  (V), wherein X is the VDA; Y is the peptide comprising the MMPproteolytic cleavage site; b is a spacer group directly or indirectlylinked to Y and wherein the spacer is selected from the group consistingof a single amino acid, amino acid sequence and a succinyl group; and cis a capping group which prevents non-specific degradation of thepeptide by enzymes other than MMPs.
 34. The method of claim 15, whereinthe compound is of formula (VI)X-a-Y-b-c  (VI), wherein X is the VDA; Y is the peptide comprising theMMP proteolytic cleavage site; a is a linker directly or indirectlyassociated with X and wherein the linker is a single amino acid or aminoacid sequence; b is a spacer group directly or indirectly linked to Yand wherein the spacer is selected from the group consisting of a singleamino acid, amino acid sequence and a succinyl group; and c is a cappinggroup which prevents non-specific degradation of the peptide by enzymesother than MMPs.
 35. The method of claim 15, wherein the compound is offormula (VII)X—Y—Z  (VII), wherein X is the VDA; Y is the peptide comprising the MMPproteolytic cleavage site; and Z is an anti-cancer agent selected fromthe group consisting of a VDA, an antimetabolite and a cytotoxic agent,wherein the VDA is selected from the group consisting ofazademethylcolchicine, colchicine, azacolchicine, N-methyldesacetylcolchicine, desacetylcolchicine, N-acetylcolchinol-O phosphate,colchicinoids, combrestatins, phenstatin, podophyllotoxins, steganacins,amphethinile, stilbenes, flavonoids, vincristine, vinblastine,vinflunine, maytansinoids, phomopson A, rhizoxin, auristatin, anddolstatin, the antimetabolite is 5-fluorouracil, and the cytotoxic agentis selected from the group consisting of anthracycline and doxorubicin.36. The method of claim 35, wherein Z is doxorubicin.
 37. The method ofclaim 35, wherein X is selected from azademethylcolchicine, colchicine,azacolchicine, N-methyl desacetylcolchicine, and desacetylcolchicine.38. The method of claim 35, wherein X and Z are selected fromazademethylcolchicine, colchicine, azacolchicine, N-methyldesacetylcolchicine, and desacetylcolchicine.
 39. The method of claim15, wherein the solid tumor is selected from the group consisting of atumor of the prostate, breast, head and neck, mouth, oral cavity, nose,trachea, lung, tongue, esophagus, stomach, small intestines, colon,liver, pancreas, gall bladder, rectum, thyroid, pituitary, adrenalglands, urinary bladder, kidney, ovary, uterus, cervix, penis, scrotum,testes, skin, brain, spinal cord, glial cells, neurons, lymphoid system,bone, cartilage, adipose tissue, smooth muscle, skeletal muscle, nervesheath, blood vessels, mesothelium and gastrointestinal stroma.
 40. Themethod of claim 15, wherein the method further comprises administering asecond therapeutic agent.
 41. The method of claim 40, wherein the secondtherapeutic agent is selected from the group consisting of cisplatin,carboplatin, cyclophosphamide, melphalan, carmustine, methotrexate,5-fluorouracil, cytarabine, mercaptopurine, daunorubicin, doxorubicin,epirubicin, vinblastine, vincristine, dactinomycin, mitomycin C, taxol,L-asparaginase, G-CSF, etoposide, colchicine, deferoxamine mesylate andcamptothecin.
 42. The method of claim 39, wherein the solid tumor is anon-small cell lung carcinoma.